Thrombin assay system

ABSTRACT

THROMBIN ACTIVITY IN BLOOD PLASMA IS QUANTITATIVELY DETERMINED BY MEASURING THE DIAMETER OF THE OPAQUE RADIAL DIFFUSION ZONE PRODUCED BY THE REACTION OF THE BLOOD PLASMA WITH UNCLOTTED FIBRINOGEN SUSPENDED IN A STABLE GEL MEDIUM.

United- States Patent 3,783,103 THROMBIN ASSAY SYSTEM Robert C. Bishop, San Gabriel, and Edward Shanbrom, Santa Ana, Calif.,"assignors' to Baxter Laboratories,

V 1 Y Inc., Morton Grove, II].

No Drawing. Continuation of abandoned application Ser.

No. 764,627, Oct. 12,1968. This 1973, Ser. No.-335,382 I 1 Int. Cl, G01n13/00, 31/14, 33/10 US. Cl, 9 t 7 195- 9 Claims ABSTRACT OF" THE DISCLOSURE Thrombinactivity in blood plasma is quantitatively determined vby measuring the diameter of the opaque radial diffusion zone produced by the reaction of the blood plasma with unclotted fibrinogen suspended in a stable gel medium. a i

,{Ifhe clotting=iof blood is of profound biological significance. It involves a complex mechanism and depends upon the'presence and activity of a number of plasma proteins; Of these proteins, fibrinogen and thrombin are of primary importance since the fundamental feature of.

application Feb. 23,

the clotting, system is the conversion of a solution of,

fibl'inogen into a rigid insoluble fibrin clot, which is normally brought about by the interaction of fibrinogen and thrombin.

The known methods for-the determination of thrombin activity generally employ reaction in a-so-called wet system as described, for example, by Iorpes et al., J Pharm and PharmacoL, vol. 10,- pp. 561 73 (1958).

,It is an object of the present invention to provide a new and improved method for the determination of thrombin activity in blood plasma and fractions thereof.

.Dther objects and advantages .Will be apparent to the person skilled in the art after reading the specification and claims hereof. I

In accordance with the present invention a stagle gel is, provided as a reaction medium for the clotting system for the determinationof thrombin activity. Unclotted fibrinogen is homogeneously suspended in the gel and then allowed to react with an unknown sample comprising blood plasma. or a fraction thereof containing thrombin for a predetermined period of time to provide an opaque radial diffusion zone of reaction product at the interface of the unknownsample and the. gel. The diam eter of the radial diffusion zone is directly proportional to the-log of'the thrombin activity in the sample being tested. The thrombin activity of an unknown sampleis determined by comparison with control samples of known thrombin activity. Y

The term stable gel is defined herein as a gel that is inert or chemically non-reactive with the fibrinogen component suspended in the gel.

In preparing the gel medium fro nabout 1.0% toabout 10.0% by weight of a gelling agent is"dissolved in a heated buffer system and then mixed with a predetermined amount of unclotted fibrinogen. Generally, from about 10 mg. percent to about 2 gm. percent and preferably about 130 mg. percent, of fibrinogcn is employed in the heated buffer system. While still hot, this mixture is poured into a low-sided flat receptacle (hereinafter also referred to as a plate) in an amount appropriate for the ICC.

size of the receptacle. The mixture is allowed to gel, and wells or cylindrical holes of approximately 1 to 10 mm. in diameter are punched or otherwise formed in the gel.

Known and unknown samples, respectively, comprising blood plasma or a fraction thereof, containing thrombin are then added to the open wells in the gel by use of a capillary pipette or similar device and the plate is incubated at about 20 to 60 C., and preferably at about 37 C., for a predetermined period of time. During this incubation, aclotting reaction occurs as the material in theLwells diffuses outwardly into the gel medium and produces an opaque radial diffusion zone of reaction product.

At theend of the incubation period, the gel is visually inspected, preferably with the assistance of a lighted magnifying viewer having a scale for measurement of the diameter of the radial diffusion zone. By comparing the diameters of the opaque radial diffusion zones produced during the incubation period by the clotting reaction in the gel in known and unknown samples, respectively, the thrombin activity in the unknown sample can be readily determined.

:In the preparation of the gel medium, any conventional gelling agent can be used, for example, gelatin, pectin, silica gel, starch, polysaccharides from seaweed such as agar, algin and carrageenin, synthetic polymeric gelling agents such as the cross-linked polyacrylamide disclosed in US. Pat. 3,046,201 and the like materials. The gelling agent preferably has the physical properties characterizing agar-agar insofar as it is readily dispersible in water and capable of forming an essentially clear hydrogel of sufficient rigidity so that the receptacle or plate containing the gel can be inverted without danger of the gel falling out. 35

A purified agarose is the preferred gelling agent employed in thepresent invention. Agarose is the neutral galactose polymer which has been separated from the agaropectin. fraction of agar by any conventional method, for example,a method suchas described in US. Pats. 3,281,409, 3,335,127 and 3,362,884. This gelling agent is preferably used at a concentration of about 2.5% by weight of the gel medium.

The .buffer'system generally employed in the gel has a pH of from about 6.0 to about 8.5 and preferably about 7.3. A preferred buffer comprises 0.154 M sodium chloride, 0.04 M imidazole and 0.02M ethylenediaminetetraacetate. Other suitable buffers which can be used are, for example, tri(hydroxymethyl)aminomethane, phosphate and barbital buffers.

The gel plate containing the unclotted fibrinogen can be overlaid with a'protective membrane, packaged by various means, ;and thereby made conveniently available for subsequent use in the determination of thrombin activity by hospitals, laboratories and other agencies and persons having a need for a simplified, yet accurate, determination of thrombin activity in blood plasma samples. The packages for these plates can be, for example, plastic film ormetal foil bags, pouches, and the like, preferably sterilized and sealed to prevent the admission of air, moisture, dirt and other contaminating materials. Suitable metal foil can be fabricated, for example, from aluminum and like metals; suitable plastic film can be fabricated, for example, from vinyl chloride and vinylidene chloride polymers and copolymers, polyvinyl chloride, polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polycarbonates, polyamides, cellulose acetate and propionate, cellulose triacetate, cellulose acetate, butyrate, ethyl cellulose, fluorocarbons, acrylic plastics such as ac-rylates and methacrylates, and polyesters such as, for example, the polyesters formed by condensation reactions between ethylene glycol and terephthalic acid.

The gel plates containing the unclotted fibrinogen can also be packaged in combination with control samples, buffer materials, capillary tubes and other components for making the complete thrombin assay.

The following examples will further illustrate the invention although the invention is not limited to these specific examples. All parts and percentages herein are on a weight basis unless otherwise specified.

EXAMPLE 1 An agarose gel plate is prepared as follows:

Preparation of reagents (A) Agarose gel plate butter-0154 M sodium chloride,

0.04 M imidazole and 0.02 M ethylenediaminetetraacetate, pH 7.30.

(B) Glycine citrated saline solution0.123 M sodium chloride, 0.020 M sodium citrate and 0.1 M glycine.

Procedure A highly purified fibrinogen solution is prepared as follows:

Pooled human plasma is frozen and then slowly thawed to 4 C. The cryoprecipitate is removed by centrifugation and dissolved in A the original plasma volume of glycine citrated saline. The pH of the solution is adjusted to 6.50 with 1.0 normal acetic acid. Then 3.5 gm. percent of polyethylene glycol 4000 (mol. wt. ca. 4000) is slowly added to the above solution and precipitation is carried out with stirring for 15 minutes at room temperature. The resultant precipitate is spun out by centrifugation and dissolved in glycine citrated saline the original plasma volume). The pH of the solution is adjusted to 6.88 with 1.0 normal sodium hydroxide and the solution is then cooled to 9 C. Glycine is slowly added to the solution to make the final glycine concentration 1.8 M and the precipitation is carried out with stirring for 45 minutes at 5 C. The resultant precipitate is spun out by centrifugation and dissolved in normal physiologic saline to produce a concentration of approximately 2.5 gm. percent fibrinogen. The fibrinogen solution is then absorbed three times with 5 gm. percent Darco G-60 charcoal. Each adsorption is carried out for thirty minutes at room temperature, the solid charcoal being removed after each process by centrifugation. The final solution is adjusted to 260 mg. percent fibrinogen by appropriate dilution with normal physiologic saline. The solution is then stored in the frozen state.

An agarose solution is prepared as follows:

2.5 gm. percent agarose is completely dissolved in the above-described agarose gel plate buifer.

An agarose gel plate containing unclotted fibrinogen is then prepared as follows:

The hereinbefore prepared agarose solution is brought to 53-56 C. and one volume of the solution is homogeneously mixed with one volume of the above-described highly purified fibrinogen solution brought to room temperature. The mixture is then poured into a plate of approximately 3 inches x'l inch x A inch deep and allowed to solidify. Six wells, each having a diameter of 2 mm. and being equidistantly spaced apart, are then punched into the gel in the unclotted plate. The plate is then capped with a protective membrane, packaged and stored at 2 to 8 C.

The agarose gel plate containing the unclotted fibrinogen is used with plasma or appropriate plasma preparations for making the quantitative determination for thrombin as follows:

A standard thrombin control sample is prepared in three concentrations, the first containing one'N.I.H.-' (National Institutes of Health) unit per rnL, the second containing 0.5 N.I.H. unit per m1. and the third containing 0.2 N.I.H. unit per m1. An aliquot from each concentration is placed in a separate well in the agarose gel plate. The unknown plasma sample to be determined is then prepared in three concentrations of 50% and 20%, respecttively, by volume of the plasma in the unknown sample. An aliquot from each concentration is placed in a separate well in the agarose gel plate. The plate is then incubated at 37 C., preferably in a moist chamber, for approximately 2 /2 hours, or until opaque reaction zones appear for all control sample concentrations. The diameter of each reaction zone is measured under the eyepiece of a Hyland Immuno-Plate Viewer or under a dissecting microscope with a stage micrometer or eyepiece reticle. The log of the control sample concentrations versus the reaction zone diameters of the respective control samples is plotted. The thrombin activity of the unknown sample can then be determined by reference to the plot formed by the control samples.

Various other examples can be devised by the person skilled in the art without departing from the spirit and scope of the invention defined herein. All such further examples are within the scope of the invention as defined in the appended claims.

What is claimed is:

1. An agarose gel plate for the quantitative determination of thrombin activity in blood plasma and fractions thereof comprising a plate and an agarose gel medium containing from about 10 mg. percent to about 2 gm. percent unclotted fibrinogen homogeneously suspended therein.

2. A method for the quantitative determination of thrombin activity in blood plasma and fractions thereof comprising reacting said blood plasma or fraction thereof with unclotted fibrinogen homogeneously suspended in a stable gel medium at a concentration of from about 10 mg. percent to about 2 gm. percent for a predetermined period of time to produce an opaque radial diffusion zone of reaction product, measuring the diameter of said radial diffusion zone and comparing with control samples of known thrombin activity, said blood plasma or fraction thereof being allowed to come into contact with said fibrinogen for reaction therewith by diffusion from a well in the surface of said gel on a plate.

3. The method of claim 2 in which the gel medium contains from about 1.0% to about 10% by weight of a gelling agent.

4. The method of claim 3 in which the gelling agent is agarose.

5. The method of claim 4 in which the gel medium contains about 2.5% by weight of agarose.

References Cited UNITED STATES PATENTS 3,482,943 12/1969 Csizmas et al. 103.5 R X OTHER REFERENCES Jorpes et al.: The Journal of Pharmacy andPharmacology, vol. 10, pp. 561-573 (1958).

A. LOUIS MONACELL, Primary Examiner I. R. HOFFMAN, Assistant Examiner US. Cl. X.R. 

